deposited in the Protein Data Bank, Brookhauen National Laboratory,. The atomic ..... Banner, D. W., Bloomer, A. C., Petsko, G. A,, Phillips, D. C., Pogson, C. I.,.
THE JOURNAL OF BIOLOGICAL CHEM~S~Y Vol. 269, No. 33, Issue of August 19, pp. 20811-20814, 1994 0 1994 by The American Societyfor Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Communication Crystal Structure, at2.6-A Resolution, of the Streptomyces lividans Xylanase A, a Member of the F Family of P-1,4-~-Glycanases* (Received for publication, May 31, 1994, and in revised form, June 22, 1994)
Urszula DerewendaS, Lora SwensonS, Ruth Green$, Yunyi WeiS, Rolf Morosolil, Francois Shareckl, Dieter Kluepfelln, and Zygmunt S. Derewendatll From the medical Research Council Groupin Protein Structure and Function, Departmentof Biochemistry, University of Alberta, Edmonton, Alberta T6G2H7 and the $Centre de Recherche en Microbiologie Appliquee, Universitk d u Q d b e c ,Znstitut Armand-Frappier, Ville de Laval, Que‘bec H7N 423, Canada
(4). acid, and several speciesof plants contain acetylated xylan Xylanases show considerable potential as bio- or prebleaching agents in pulp and paper industries allowing for the elimination of polluting chlorine (5). Amino acid sequence analyses strongly suggested that t h e F and G families of glycanases are structurally different (1,2). To date, three crystal structures of enzymes belonging to the G family have been reported: from Bacillus pumilus (61, Pichoderma harzianum (71, and Bacillus circulans (7, 8). These enzymes share a tertiary fold consisting of three P-sheets and an a-helix. The active siteis i n a cleft at the intersection of two sheets (8). Two glutamates, 78 and 172 (B. circulans sequence), have been found to play key catalytic roles (8). Previously we reported cloning and characterization of three different xylanases fromStreptomyces liuidans (9-12). The enzymes denoted XlnB’ and XlnC belong to the G family, while XlnA, a 47-kDa single chain polypeptide of 436 amino acids representative (GenBank accession number M64551, 1991)a is of t h e F family. Three successful crystallizations of catalytic domains of F family xylanases have been reported so far (13is available to date.We now ofE ) , but no structural information report the crystal structure of the 32-kDa catalytic domainof the S. liuidans x l n A (henceforth denoted kDa), refined at 2.6-a resolution, the first description of a three-dimensional structure of an F family glycanase.
The crystal structure of the 32-kDa catalytic domain the Streptomyces Zividansxylanase A was solvedby molecular isomorphous Feplacement methods and subsecrysquently refined at 2.6-Aresolutionto a conventional tallographic R factor of 0.21. Thisis the first successful structure determination of member a of the F family of MATERIALSANDMETHODS endo-/3-1,4-~-glycanases. Unlike the recently determined xylanases of the G family (Wakarchuk, W. W., Campbell,R. Enzyme Expression and Purification-Cellulolytic enzymes are typiL., Sung, W. L., Davoodi, J., andYaguchi,M. (1994)Protein cally made up of an N-terminal catalytic core and a C-terminal subSei. 3, 467-475),where the catalytic domains have a strate binding domain; most crystallographic studies focus on the cataunique /3-sheet structure, the 32-kDa domain of the S. lytic domains. The x l n A gene contains at its C terminus a reiterated zividuns xylanase Ais folded into a complete (d/3)e barrel, amino acid sequence of37 residues (316-353 and 400-436). Similar the first such fold observed among /3-1,4-~-glycanases. repeats were describedin glucanases as substrate binding domains (16, 17), and their deletion did not affect the hydrolytic function of the The active site is located at the carbonyl end of the /3 enzyme (18). Truncated X n lA ,, was prepared using standard procebarrel. The crystal structure supports earlier the assign- dures (19) and expressed in S. Ziuidans IAF 10-164 (20). The plasmid ment of Glu-128 and Glu-236 as the catalytic amino acids pIAF18 was digested with SstI (nucleotide 1297)and religated on itself. (Moreau,A, Roberge,M., Manin,C., Shareck, F., Kluepfel, Transformants harboring the 5.4-kilobasepair fragment in the opposite D., and Morosoli,R. (1994) Biochem.J., in press). orientation were screened onsolid agar medium containing 0.15%
XlnA,
oat spelt xylan (Sigma) covalently linked to Remazol Brilliant Blue (Aldrich) for the appearance of fuzzy substrate clearing zones around the colonies (20, 21).Presumptive clones were analyzed for production Cellulases and xylanases are classified into11distinct fami- of truncated xylanase. The clone S. liuidans IAF19 was used for the lies (1-3). The ~-1,4-~-xylan-xylanohydrolases, or xylanases overexpression of the protein as described elsewhere (11). The protein (EC 3.2.1.8), are included in two of these families, denoted F was recovered from the culture filtrates by ethanol precipitation, disand G. They hydrolyze P-l,4-~-glycosidic bonds in xylan, a com- solved in 20 mM Tris-HC1buffer, pH 8.3, centrifuged, and dialyzed ponent of hemicelluloses or low molecular weight heteroglycans overnight against the same buffer. This solution was adsorbed on a (Waters), and the associated in plant cell walls with lignin and cellulose. The semipreparative AP-215HRDEAE-HPLCcolumn protein was eluted with a linear gradient of 1M NaCl. The active fracmain chain of xylan is composed of D-xylose; branches consistof tions were pooled and dialyzed against H,O (MilliQ) and lyophilized. L-arabinofuranose and D-glucuronic or 4-0-methylglucuronic It should be noted that the SstI cleavage site occurs downstream from the first sequence repeat in the putative substrate binding domain (9). * This research was supported in part by the Medical Research Coun- Consequently, we should obtain a 39-kDa fragment. However,upon cil of Canada, a grant to the Group in Protein Structure and Function purification of the recombinant protein, SDS-polyacrylamide gel elec(toZ. S. D.), a grant from the Alberta Heritage Foundation for Medical trophoresis indicated a molecular mass of 32 kDa. This is most likelya Research (to Z. S. D.), and a co-operative research and development result of a post-translational proteolytic event. were obtained using the CrystalZizationSingle crystals of grant from NSERC (to R. M, F. S., and D. K.). The costs of publication of this article were defrayed in part by the payment of page charges. hanging drop method by dissolving the protein in distilled water (60 This article must therefore be hereby marked “aduertisement” in ac- mg/ml) and mixing it in a 1:l ratio with the stock solution of 5% polycordance with 18 U.S.C. Section 1734 solelyto indicate this fact. ethylene glycol 4000, sodium acetate buffer (100 m),pH 4.6. A 6-pl The atomic coordinates and structure factors (code IXAS) have been drop was suspended over a well of stock solution. The crystals are deposited in the Protein Data Bank, Brookhauen National Laboratory, Upton, NY ll To whom correspondenceand reprintrequests should be addressed. kDa, 32-kDa cataThe abbreviations used are: Xln, xylanase; Tel.: 514-687-5010;Fax: 514-686-5501. lytic fragment of the S. liuidans xylanase A CBHII, cellobiohydrolase 11 Medical Scholar of the AHFMR. To whom correspondence should be from T reesei; CelD, endoglucanase from C. therrnocellum; EGV, endoaddressed. Tel.: 403-492-2136;Fax: 403-492-0886. glucanase V from H.insolens.
X n lA,
Xl*,
20811
.3
20812
Crystal Structure of S. lividans XylanaseA
TABLEI Data collection, phasing statistics, and model Stereochemistry The various crystallographic parameters are defined as follows: R,1 1 Fobs- Fcale I /z 1 FobsI , where Fobsand FEQle represent respectively the observed and calculated structure factors of the i-th reflection, and the summation extends over all data; Rmerge, 1I I,- (I) I where I, is the intensity of the i-th observation and (I) is the mean intensity of the reflection; Rise, 1 I F,, - FpI E 1 FpI , the mean relative isomorphous differencebetween the data; RCullia, 1 llFpH+ FpI - FHI /X I FPH- FpI , where F, is the calculated heavy atom structure factor native protein (F,) and derivative where ( E ) is the root-mean-square lack of closure. contribution. Phasing power, (FH)/(E),
/u,,
X-ray data collection
2.60Nominal resolution (A) Unique 88.8Overall completeness (%) Inner shell Outer shell (I/dI)) Outer shell 4.1 Redundancy 7.4 R , e (%) Rim?%) Number of sites
Native
Platinum
2.59 8479 92.9 100.0 (20.0 8, - 2.75A) 56.1 (2.75 A - 2.59A) 52.0 63.1 9.08 (2.75 A - 2.59A) 8.2 6.7
6769
Iodine
7980
A)
97.4 (20.0 A - 2.90 A) 41.6 (2.90A - 2.73 A)
96.2 (20.0 A - 2.76 51.1 (2.76A - 2.60 A) 37.8 8.34 (2.76 A - 2.60 A)
8.00
12.6 1
(2.90 A - 2.73 A) 30.8 6
Overall phasing results
R,,,i, (acentric) RcuIli,(centric)
0.89
0.71 0.85
Phasmg power (acentric) Phasing power (centric) Figure of merit ( m ) Acentric Centric
0.90 1.1
1.8 1.2
0.8
0.52 0.74
Figure of merit in resolution shells before and after SQUASH (28)
Resolution 3.8 4.3 (A) 5.3 6.3 Figure of merit m.i.r. 0.65 0.60 0.81 0.84 (SQUASH) m.i.r. 0.40 0.53 0.65 0.92
3.5
0.12 0.67 0.920.74
0.85 0.76
0.28 0.39 0.55 0.46 0.80
0.51
Refinement and model stereochemistry
X-ray data used for refinement Resolution limits 10 - 2.6 A Minimum I / u ( I ) 0 Root-mean-square deviation from ideal stereochemistry (target u in parentheses) 1-2 distances (0.02) 0.011 A 1-3 distances (0.04) 0.045 A Planar groups (0.02) 0.012 Chiral volumes (0.10) 0.095 A3 Planar peptides (20)’ 3.2 Secondary structure analysis according to PROCHECK (47) Residues favoredin most regions (%) 86.3 Residues in additional allowed regions (%) 13.4 Non-glycine amino acids in disallowed regions 0
A
p
orthorhombic, space group P2,2,2,,a = 69.5A, b = 46.5A, c = 85.5A . The value of V,,, (231,assuming one molecule in the asymmetric unit, is2.15 A3~a. Data Collection-All diffraction data were recordedusing a Siemens rotating anode generator operated at 40 kV and 90 mA, equi ped with a CuKa target,a graphite monochromator (A = 1.5418 ), and a Siemens X1000 area detector. Data were processed and reduced using XENGEN (24)and the CCP4 program suite (SERC Daresbury Laboratory, United Kingdom). Structure Solution-Tbro heavy atom derivatives were prepared by soaking native crystals under the following conditions: platinum, 7 mM chloro-(2,2‘:6’,Z”4erpiridine)platinum I1 chloride (25)for 60 h; iodine, 10 mM N-iodosuccinimideanhydride for 9 h. All subsequent calculations were carried out using the CCP4 suite, unless stated otherwise. Asingle platinum site was identified using SHELXS-90 (26)and used to calculate single isomorphous replacement anomalous scattering phases; the latter were used in difference Fourier calculations, which revealed six iodine sites in the second derivative. The refinement of all heavy atom parameters and calculation of phases were carried out using MLPHARE (27) in the CCP4 implementation. Density modification using SQUASH (28)improved the quality of phasing further and and allowed for a calculation of a fully interpretable electron density map. Model building, using “ 0(29),was significantly facilitated by the recmolecule exhibits a fold characognition, early on, that the X l & 2 teristic of an barrel. Amodel consisting of all but the 5 N-terminal and 16 C-terminal residues was refined using XPLOR (301,and the
8:
resulting difference density map revealed the missing C-terminal amino acids. Refinement-Refinement of the model was carried out by iterative rounds of restrained crystallographicrefinement using XPLOR (29)and PROLSQ (CCP4) with intervening manual revisions of the structure using “ 0(28) on an SGI Indigo2Extreme and Evans & Sutherland ESV 20 systems. Both positional and thermal parameters were refined, albeit the latter were strongly restrained due to the relatively low resolution of the data.Over 200 water molecules wereidentified on the basis of positive differenceelectron density and stereochemical criteria. The refinement converged with the standard crystallographic R factor of 0.21 and good protein stereochemistry. RESULTS AND DISCUSSION
The resultsof x-ray data collection, phasing, refinement, and the stereochemistry of the final model are documented in Table I. The final model contains 295 contiguous amino acids beginning with the Leu-5 and ending with Ala-299. Some residual lnA,kDa fragment density beyond Ala-299 indicates that the X may contain few more residues, albeit disordered in thecrystal structure. Of the 200 refined solvent molecules, over 100 retained good hydrogen bonding patterns. The molecule exhibits a tertiary fold of a typical barrel, firstidentified in triose-phosphate isomerase (31).
X n lA ,,
Crystal Structure of S. lividans Xylanase A
20813
-1
FIG.1. A schematic representation of the three-dimensional structure of the molecule of The P-strands forming the barrel are blue, and the eight main helices are yellow. The short red helix in the foreground is the N terminus,and the strand seen behind it is strand 1. The two shortpurple helices occur within complex turns (white)on the top (substrate binding) faceof the molecule. The C terminus is at the bottom of the yellow helix immediatelyto the left of the N-terminal helix.The secondary structure elements are definedas follows. P-Barrel strands: p l (15-201, p2(40-44), /33(77-84),p4(121-127), p5(166-171), p6(200-208), @7(231-240), p8(261-266); outer barrel helices:- a1(20-37), a2(62-73),a3(98-116),a4(150-161),a5(180-195), a6(218-226),a7(244-254),a8(291-298); additionalshorthelices: a0 (N-terminal)(6-9), a l ' (49-52), a2' (91-94).
FIG.2. A schematic diagram illustrating thestructure and hydrogen bonding of the f3-barrel.Typical P-sheet hydrogen bonds are shown as urrows pointing fromthe donor main chain amidesto carbonyl oxygens. Atypical bonds are indicated dashed by urrows.0, residues in the a-helical conformation; @, residues in the aL (left-handed helix) conformation;@, residues in the "a" regionas defined elsewhere(47).
Although the details of the mechanism of the hydrolytic cleavage of glycosidic bonds by cellulases and xylanases may vary7the hen egg white lysozyme paradigm (42) with Asp-52 and Glu-35 involvedin catalysis is a common theme. Variations include the presence of 2 aspartates, as in EGV (41)and CBHII (38), or 2 glutamates, as in G family xylanases(8). In contrast to inverting enzymes, suchas CBHII, EGV and CelD, boththe G and F families of xylanases hydrolyze the P-1,4-glycosidic bond with a retention of configuration at C-1 (431, as does Fig. 1shows a schematic view of the overall polypeptide chain lysozyme. Thereactionproceedsvia a doubledisplacement fold. Seen from the side the molecule has a "salad bowl" shape. mechanism, whereby a general acidhucleophilic attack on the The face of the molecule onthe carbonyl sideof the P barrel (or anomeric center could result either in an oxycarbonium ion the top face)has a larger radius, -45 A, due to a more elaborate architecture of the P-a loops. The bottom face, consisting of stabilized by a nearby carboxylate or in a covalent glycosyl enzyme intermediate. In either case, a nucleophilic water molsimple a-p turns, hasa radius of approximately30 A. The ( d p ) , ecule completesthe pathway by generating reaction products. barrel constitutes the most common tertiary fold among enzymes (32-34). Individual barrels vary significantly, with cross-The oxycarboniummechanism is generally accepted forlysections ranging from nearly circular, as in glycolate oxidase sozyme with Glu-35acting as a general acid and Asp-52 stabi(351, to quite elliptical, e.g. triose-phosphate isomerase, where lizing the oxycarbonium ion (44). In theG family xylanases,it the short and long diameters are 11.5 and 16.5 A. However, is suggested that Glu-78 and Glu-172 (B. circulans notation) with a notable exception of enolase, which contains one anti- act as the nucleophile and general acid, respectively(8). In XI&, 2 residues have been implicated in catalysis by parallel strand (361, they are always made of eight parallel site-directed mutagenesis studies (45). Replacementof Glu-128 P-strands, tilted with respectto each other by about -35 "C and and Glu-236 by isosteric glutamines completely abolished enwith a shear numbe? of 8 (37). zymatic activity in the mutant enzymes, both against xylan barrel is atypical. It is the most elliptical barThe In a fashion typical of and p-nitrophenyl-~-l,4-~-cellobioside. and rel kn:wn, with the short and long axes approximately 9.5 all (a@), barrels, Glu-128 and Glu-236 are located at the car17.5 A, respectively. In addition, strands 3, 4, and 7 contain bulges with5 amino acids adoptingright- or left-handed a-hel- bonyl ends of strands 4 and-7, respectively, within a shallow ical conformations, an unusual distortion that affects the reg- depression approgmately 8 A deep. Their Ca atoms are sepaularity of hydrogen bonding (Fig. 2).A high resolution refine- rated by about 13A, while the carboxylate carbonsby 7 A. This is very similar to lysozyme, where the latter distance is 7.3 A. structure will address all pertinent ment of the W e conclude that the substrate polysaccharide probably binds stereochemical details. in the shallow groove alongthe top faceof the molecule (Fig.3) Cellulases and xylanases encompass a wide variety of enand that glutamates 128 and 236 indeed constitute the catazymes (3), few of which have been characterized by x-ray cryslytic residues.On the basis of hydrophobicity profileanalysis of tallography. Theknown cellulase structures are: cellobiohydroX l n A 3 2 k D a 7 it was postulated that Glu-128 functionsas a genlase I1(CBHII)from Dichoderma reesei (38), a related endocellulase from Thermomonospora fusca, (391, both repre- eral acid (45). The crystal structure corroborates these conclusions. Glu-236is located in a cluster of polar and charged resisenting the B family of glycanases (1);Clostridium thermoceldues in close proximity of Arg-139 and His-86. Moreover, it Zum endoglucanaseCelD (40), a member of the E family (2);and accepts two hydrogen bonds to its side chain carboxyl group the Humicola insolens endoglucanaseV (EGV)(41),from the K family(3).None of these structures is a typical barrel. from Ne2 of His-207 and N62 of Asn-170. There is little doubt that this residue is in an ionized state. In contrast, Glu-128 The uniquep-sheet fold of the three known G family xylanases N.52 of Gln-205, whilethe Oel has already been mentioned. Thus, the MI&,, structure is accepts one hydrogen bond, from to a water molecule, like Glu-35 in atom is hydrogen-bonded the first example of a p-l,4-~-glycanasewith an (dP),barrel lysozyme. fold. Another interesting observation relates to the function of Asn-173. The N173D mutation alters themode of action of the Shear number is the residue advance going.aroundthe barrel to return to the starting amino acid. enzyme on xylopentaose, so that instead of cleaving the second
=I&,
XlnA,,
Crystal Structure of S. lividans Xylanase A
20814
6. Katsube, Y., Hata, Y., Yamaguchi, H., Moriyama, H., Shinmyo,A,, and Okada, H. (1990) in Protein Engineering: Protein Design in BasicResearch, Medicine and Industry (Ikehara, M.,ed) pp. 91-96, Japan Scientific Societies Press, Kobe, Japan 7. Campbell, R. L., Rose, D., Wakarchuk, W., To, R., Sung, W., and Yaguchi, M. (1993) Found. Biotech. Ind. Ferment. Res. 8,63-72 8. Wakarchuk, W.W., Campbell, R. L., Sung, W. L., Davoodi, J., andYaguchi, M. (1994) Protein Sei. 3,-467475 9. Shareck, F., Roy, C., Yaguchi, M., Morosoli, R., and Kluepfel, D. (1991) Gene (Amst.) 107,7542 10. Morosoli, R., Bertrand, J. L., Mondou, F., Shareck, F., and Kluepfel, D. (1986) Biochem. 239.587492 ." .~ . J. . ~~. 11. Kluepfel, D., Vats-Metha, S., Aumont, F., Shareck, F., and Morosoli, R. (1990) Biochem. J. 287,4550 12. Kluepfel, D., Daigneault, N., Morosoli, R.,and Shareck, F.(1992) Appl. Micmbiol. Biotechnol. 36, 626-631 13. Bedarkar, S.,Gilkes, N. R., Kilbum, D. G., Kwan, E., Rose, D. R., Miller, R. C., Jr., Warren, R. A. J., and Withers, S.G. (1992) J. Mol. Bwl. 228,693-695 14. F'ickersgill, R.W., Jenkins, J. N., Scott, M., Connerton, I., Hazlewood, G. P., and Gilbert, H. J. (1993) J. Mol. Biol. 229,246-248 15. Souchon, H., Spinelli, S., Beguin, P., a n d h a r i , P. M. (1994) J. Mol. Biol. 235,
L
~
FIG.3. The active site with selected amino acids. view down the barrel axis. The 2 active site glutamates (Glu-128 and Glu-236) are colored red;Am-173 is shown in green. The p-strands of the barrel and the loops are shown in blue. AU helices are in yellow. The substrate is most likely to bind fromleft to upper right, along the short axis of the barrel, with the reducing end pointing to Asn-173.
and the third p-1-4- linkages (numbered from the reducing end) withthe same frequency, the second linkage is hydrolyzed preferentially (20). Fig.3 shows the location of Asn-173 within the active site. The wild type mode of action on xylopentaose may indicate the presence of six principal binding subsites, so that xylopentaose could either bind A(l)-B(2)-C(3)-D(4)-E(5), or A(2)-B(3)-C(4)-D(5)-E(6).3 In both cases, the cleavage would occur between subsites 3 and 4.The proposed role of Asn-173 in the enzymatic mechanism (20) is consistent with this amino acid's involvement in subsite 6. Given the relative positions of Glu-128, Glu-236, and Asn-173, and based on the lysozyme paradigm (441, we conclude that thecrystal structure supports this conclusion, and that the location of Asn-173 (Fig. 3) points to the reducing-end of the bound xylan. The N173D mutant would favor theA(l)-B(2)4(3)-D(4)-E(5)binding mode, leading to the preferential cleavage between C and D subunits. Further research focusing on mutational, crystallographic, and kinetic studies of the enzyme, currently in progress, w l l i hopefully lead to full elucidation of the mechanism of the F family glucanases.
7
-
-
~~~
1348-1350 16. Faure, E.,Belaich, A, Bagnara, C., Gaudin, C., and Belaich, J.-P. (1989) Gene (Amst. ) 84,39-46 17. Grepinet, O., Chebrou, M.-C., and Beguin, P. (1988) J. Bacteriol. 170, 4 5 8 Z 4588 18. Hall, J., Hazzlewood, G. P., Barker, P. J., and Gilbert, H. J. (1988) Gene (Amst.) 69,29- 38 19. Hopwood, D. A., Bibb, M. J., Chater, K C., Kieser, T., Bruton, C. J., Kieser, €€ M., Lydiate, D. J., Smith, C. P., Ward, J. M., and Schrempf, H. (1985)Genetic
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Acknowledgment-We thank Janusz Chlebek for preparing Fig. 2. REFERENCES 1. Henrissat, B., Claeyssens, M.,Tomme,P.,Lemesle,M., and Mornon, J. P. (1989) Gene (Amst. 81.83-95 2. G&es, N. R., Henrissat, B., Kilbum,D. G., Miller, R. C., and Warren, R. A. J. (1991) Microbiol. Reu. 55, 303-315 3. Henrissat, B., and Bairoch, A. (1993) Biochemistry 293,781-788 4. Biely, P. (1985) %rids Biotechnol. 3,286-290 5. Senior, D. J., Hamilton, J., Bernier, R. L., and du Manoir, J. R. (1992) Tappi. J. Nou 125-130
Letters denote the xylose subunits, and numbers denote the binding subsites, in the direction from the non-reducing to the reducing end.
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